In order to genetically manipulate key proteins involved in the autonomic regulation process, we had to develop a technique for the culture of rabbit Sinoatrial node cells, as its impossible to do so in freshly isolated SANC. We have been able to obtain stable adult rabbit cultured SANC (c-SANC), to characterize their properties, and have successfully overexpressed proteins in c-SANC via adenovirus directed acute gene-transfer technique. Our results show that on the first day of primary SANC culture, most of the cells tend to spread out and could stay alive for up to 8 days, a period which would allow us to introduce exogenous proteins into c-SANC. By immuno-staining, we detected essential proteins involved in autonomic regulation in c-SANC, including type 2 sarcoplasmic reticulum Ca2+ release channel, i.e. type 2 ryanodine receptors (RyR2), L-type Ca2+ channel, hyperpolarization-activated cyclic nucleotide-gated channel 4, phospholamban (PLB), Sarco/Endoplasmic Reticulum Ca2+-ATPase 2a and Sodium-Calcium exchanger. At 34 0.5 degrees C, c-SANC generate spontaneous, rhythmic action potentials (AP), but at a level (1.35 0.02 Hz, n=803, 2 to 8 days into culture) significantly slower than f-SANC (2.79 0.04 Hz, n=203, p<0.001). c-SANC also generate AP triggered global Ca2+ release transients, and spontaneous Local Ca2+ Releases (LCR) beneath the plasma member during diastolic depolarization, just prior to the global Ca2+ transients. Some characteristics of AP, Ca2+ transients and LCR in c-SANC are similar to those from f-SANC, but with critical difference (e.g. LCR period, AP Ignition period and the kinetics of AP and Ca2+ transients) which are strongly correlated with the different spontaneous AP firing rate. It is well documented that the peptide inhibitor of protein kinase A (PKA), PKI, can dramatically reduce or stop the beating rate of f-SANC. We hypothesized that the relatively low beating rate of c-SANC, is possibly due to the down-regulated PKA signaling in the cultured cells. Indeed, acute stimulation of beta-adrenergic receptors with 1 microMolar isoproterenol (ISO) for 10 min accelerates AP and Ca2+-transient kinetics, reduces the LCR period, and accelerates the AP firing rate to a similar maximum in c-SANC (3.34 0.05 Hz, n=150) and f-SANC (3.55 0.06 Hz, n=126). In addition, we observed that the phosphorylation level of RyR2, which is indexed by the fluorescence density of phosphorylated RyR2 at Ser2809 normalized by its own total RyR2 fluorescence density, is substantially lower in c-SANC (1.32 0.06, n=47) than in f-SANC (1.66 0.15, n=24, p<0.01). While acute ISO stimulation raises the RyR2 phosphorylaiton at Ser2809 to a similar level in both cell types, PKI treatment reduces the phosphorylation level. More specifically, the phosphorylation level of PLB at Ser16, a PKA specific site, is also significantly lower in c-SANC than f-SANC. Similarly, ISO acute stimulation increases and PKA inhibition by PKI decreases PLB phosphorylation at Ser16 in both cultured and freshly isolated SANC, supporting the interpretation that PKA signaling is down-regulated in cultured SANC compared with freshly isolated SANC. Whats the mechanism underlying the PKA down-regulation in cultured pacemaker cells? Based upon the above data and the fact that the activation of pertussis toxin (PTX)-sensitive Gi signaling is involved in the beating rate reduction of f-SANC, we measured the protein expression level of type 2 regulator of G protein signaling (RGS2), which functions as a powerful negative regulator of PTX-sensitive Gi signaling. As we expected, the protein level, indexed by the immuno-labeling density along the cell membrane, is substantially lower in 2 day cultured SANC (149.9 4.0, n=100) than in f-SANC (201.9 6.0, n=88, p<0.001). 2 hours incubation of 1 microMolar ISO enhances the staining density of RGS2 and PKI completely inhibits ISOs effect. Functionally, over-expression of RGS2 via adenovirus directed acute gene-transfer technique increases the spontaneous beating rate of cultured SANC from 1.35 0.05 Hz (n=91) to 1.86 0.05 Hz (n=50, p<0.001), which is 66% of f-SANCs AP firing ate. This effect is not because of adenovirus infection, as introducing the green fluorescent protein (GFP) into c-SANC via the same technique, does not affect the cell beating rate, and there is no correlation between AP firing rate and GFP expression level. Furthermore, when cultured SANC were treated with 0.4micrograms/ml PTX overnight, the spontaneous beating rate is boosted to 2.38 0.11 Hz (n=45), 85% of f-SANCcs AP firing rate. Partial rescue of c-SANCs AP firing rate by PTX treatment or RGS2 overexpression indicate that a reduction in PKA-dependent Ca2+-cycling protein phosphorylation that is Gi-dependent is involved in prolongation of LCR period and reduced spontaneous AP firing rate of c-SANC, and that this deficit can be reversed by pharmacologic or genetic manipulation. We concluded that adult c-SANC provide a reliable model to study the autonomic regulation by acute genetic manipulation of key proteins. More importantly, understanding the difference between freshly isolated and cultured SANC itself broaden our view of autonomic regulation. We also successfully over-expressed some Ca2+ regulatory proteins in c-SANC, including wild type and mutant Ca2+ / calmodulin-dependent kinase IIgammaC (a multifunctional Ca2+ dependent kinase), Ht31 (a peptide that binds the PKA regulatory subunit type II (RII) and competes with endogenous A-kinase anchoring protein for RII binding, thus interrupts AKAP mediated PKA anchoring) and its inactive form Ht31p. These projects are still in progress, and should lead to several publications in the near future.